NAD and NADP are naturally occurring energy sources in cells. They are important cofactors to numerous enzymatic reactions. They are also substrates for NAD(P)ases which catalytically break down these products into nicotinamide and ADP-ribose or ADP-ribose phosphate. In addition, NAD is also a substrate for poly (ADP-ribose) polymerase and mono (ADP-ribose) transferases important in DNA repair, apoptosis, differentiation and signal transduction (Althaus and Richter, ADP-ribosylation of Proteins: Enzymology and Biological Significance, Springer-Verlag, Berlin, 1987; Satoh et al., Biochemistry 33: 7099-7106, 1994; Kaufman et al., Cancer Research 53: 3976-3985, 1993; Lindahl et al., TIBS 20: 405-411, 1995; Gilman AG, Cell 36: 577-579, 1984; Lee and Iglewski, Proc. Natl. Acad. Sci. USA 81: 2703-2707, 1984).
There are no known useful functions for the NAD(P)ases in cells: A group of NADases also possess ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities (Galione and White, Trends in Cell Biol. 4:431-436, 1994; Lee et al, Biochemie 77: 345-355, 1995).
These enzymes were first described in sea urchin eggs and they have been purified (Rusinsko and Lee, J. Biol. Chem. 264: 11725-11731, 1989; Lee and Aarhus, Cell Regul. 2: 203-209, 1991; Hellmich and Stumwasser, Cell Regul 2:193-203, 1991) and cloned (Glick et al., Cell Regul 2: 211-218, 1991). The NAD hydrolyzing properties are coupled to synthesis of cyclic ADP-ribose (cADPR) (Lee et al. J. Biol. Chem. 264: 1608-1615, 1989), which has been shown to have secondary messenger properties involved in the induction of Ca2+ release (Galione and White, Trends in Cell Biol. 4:431-436, 1994; Lee et al, Biochemie 77: 345-355, 1995).
Some of these enzymes are bifunctional having both ADP-ribosyl cyclase and cADPR hydrolase activities, and consequently can both synthesize cADPR or hydrolyze cADPR to ADP-ribose (Kim et al., Science 261:1330-1333, 1993; Lee et al, Biochemie 77: 345-355, 1995; Galione and White, Trends in Cell Biol. 4:431-436, 1994). One of these bifunctional enzymes is the CD38 ectoenzyme in mammalian B- and T-lymphocytes and myeloid cells (Lund et al., Immunol. Today 16: 469-473, 1995) and in insulin secretory human xcex2-cells (Okamoto et al., Biochimie 77:356-363, 1995). The CD38 receptor has been shown via Ca2+-release to be involved in the regulation of B cell proliferation (Howard et al., Science 262:1056-1059, 1993; Kumagai et al., J. Exp. Med. 181: 1101-1110, 1995), in T, NK and plasma cell regulation (Funaro et al., J. of Immunology 145: 2390-2396, 1990) and it is upregulated in HL-60 cells stimulated to differentiate by retinoic acid (Kontani et al., J. Biol. Chem. 268:16895-16898, 1993). Earlier it has also been shown (Hemmi and Breitman, Biochem. Biophys. Res. Com. 109: 669-674, 1982) that treatment with retinoic acid induced NADase activity in the same cell line, indicating a strong coupling between NADase- and CD38/cyclase activity.
NADase/cyclase is an ectoplasmically located cellular enzyme. Hence, when cells are supplied with NAD or NADP they can serve both (i) as substrates for the production of cyclic ADP-ribose, imbalance Ca2+ and induce cytotoxicity and (ii) as a catabolic source for the generation of nicotinamide which can increase tumor blood flow. Both the apoptotic cytotoxicity and enhanced tumor blood flow are well documented mechanisms by which conventional radio- and chemotherapies can be sensitized (Pero et al, Cancer Det. Prevent. 22(3): 225-236, 1998; Horsman, Acta Oncologica 34: 571-587, 1995). It follows then that NAD and NADP are novel antitumor drugs because they can combine two important mechanisms of action into the same compound by acting as a metabolic prodrug.
Further to this invention is the disclosure that ADP-ribose cyclases such as CD38 are important metabolic sites for the expression of the desired pharmacological effects of NAD. Therefore, agents that can serve as precursors or metabolic products of NAD and NADP judged by their abilities to interact with cyclases should also be considered appropriate to this invention. For example, but not limited to, there are the metabolites and substrates such as cyclic ADP-ribose, ADP-ribose, 2xe2x80x2 and 3xe2x80x2 deoxy NAD, 2xe2x80x2 and 3xe2x80x2 deoxy ADP-ribose, NADH, and NADPH (Gailone and White, Trends in Cell Biology 4: 431-436, 1994; Ashamu et al, Biochemistry 36: 9509-9517) as well as other affinity binding agents to ADP-ribose cyclases (NADases) such as 3-acetyl pyridine adenine dinucleotide, 3-acetyl pyridine, nicotinamide mononucleotide, benzamide and 3-aminobenzamide (Olsson et al, Biochem. Pharmacol. 45:1191-1200, 1993).
This invention discloses a method by which agents such as NAD and NADP, or its metabolic precursors, products, and competitive substrates which agonize/antagonize ADP-cyclase and produce or inhibit cyclic ADP ribose and in turn imbalance intracellular calcium are useful in inducing or inhibiting apoptosis or clonogenic cytotoxicity in tumor, microorganism, or normal cells. In this regard, tumor cells for example having a high proliferative capacity are forced into a pathway of differentiation and eventual apoptotic cell death. Because the large majority of normal tissues of the body are not undergoing cell division, a therapeutic gain of killing tumor tissues over normal tissues is realized. The useful method is comprised of the steps of administering by an appropriate route (e.g. intravenous, oral, intramuscular) to a patient having a tumor, NAD or NADP at a dose sufficient to elevate cyclic ADP ribose, imbalance calcium and induce apoptotic cytotoxicity, so that proliferating tumor cells are inhibited to grow.
In another aspect, this invention teaches that treating individuals having tumors with therapeutic doses of NAD or NADP or an appropriate analog will not only kill tumor cells by apoptosis, but the enzymatic product of this process, cyclic ADP(P)ribose, also results in the production of another well known radio- and chemosensitizer, nicotinamide. Consequently, combining NAD or NAD(P) with conventional radio- or chemotherapies permits sensitization by both the apoptotic as well as nicotinamide cytotoxic mechanisms (i.e. increasing tumor blood flow; Horsman, Acta Oncologica 34: 571-587, 1995). Hence, the method taught by this invention may be augmented by co-administering to the tumor patient traditional radio- or chemotherapies in combination with useful formulations of NAD and NADP as a sensitizing adjuvant therapy.
In still another aspect, this invention teaches that NADases or ADP-ribose cyclases could also be viewed as a transport mechanism for internalizing into cells phosphorylated adenosine-containing precursors for eventual use in DNA synthesis. It is known that the ADP-ribose moiety of NAD becomes selectively taken up by cells when supplied with exogeneous NAD (Pero et al, In: ADP-ribose transfer reactions. Biological significance, Eds. M. K. and E. L. Jacobson, pp 378-385, Springer, N.Y., 1989). Stimulating ATP synthesis selectively would no doubt imbalance nucleotide pools and provide opportunities for antimetabolite therapy (Harrup and Renshaw, Antibiotics Chemother. 28: 68-77, 1980). Adenosine compounds, some of which can imbalance nucleotide pools by inhibition of adenosine deaminase such as coformycin and deoxycoformycin, have been used for the successful treatment of neoplastic, fungal and parasitic diseases (Harrup and Renshaw, Antibiotics Chemother. 28: 68-77; U.S. Pat. Nos.: 5,180,714; 4,997,818; 5,679,648). Here I disclose that adenosine-containing analogs that can bind to the ectoplasmically located NADases (ADP-ribose cyclases) and become internalized into the cell and imbalance nucleotide pools have utility in the treatment of neoplastic, fungal and parasitic diseases.